Isaacs and Lindenmann reported in 1957 that when chicken is infected with influenza virus A, a viral replication inhibitory factor designated interferon is produced (Isaacs, K and Lindenmann, J. Proc. R. Soc. Lond., B147:258–267, 1957).
Human interferons are cytokine proteins which inhibit in vivo immune response or viral replication and they are classified as interferon alpha (IFNα), interferon beta (IFNβ) and interferon gamma (IFNγ) according to cell types producing them (Kirchner, H. et al., Tex. Rep. Biol. Med., 41:89–93, 1981; Stanton, G. J. et al., Tex. Rep. Biol. Med., 41:84–88, 1981).
It is well-known that these interferons work together to exert synergic effects in the manifestation of anti-viral, anti-cancer, NK (natural killer) cell activation and marrow cell inhibition activities (Klimpel, et al. J. Immunol., 129:76–78, 1982; Fleischmann, W. R. et al., J. Natl. Cancer Inst., 65:863–966, 1980; Weigent, et al., Infec. Immun., 40:35–38, 1980). In addition, interferons act as regulatory factors of the expression, structure and function of genes in the cell, and show a direct anti-proliferating effect.
IFNα is produced when leukocyte is stimulated by B cell mitogen, virus or cancer cells. Up to now, there have been reported genes that encode more than 20 species of interferons, each comprising 165 or 166 amino acids.
IFNα used for early clinical tests were obtained from buffy coat leukocyte stimulated by Sendai virus and its purity was only less than 1% (Cantell, K. and Hirvonen, Tex. Rep. Biol. Med., 35:138–144, 1977).
It has become possible to produce a large quantity of IFNα having biophysical activity by gene recombinant techniques in the 1980' (Goedell, D. V. et al., Nature, 287:411–416, 1980). Clinical tests using the recombinant hIFNα have shown that it is effective in treating various solid cancers, particularly bladder cancer, kidney cancer, HIV related Kaposi's sarcoma, etc. (Torti, F. M., J. Clin. Oncol., 6:476–483, 1988; Vugrin, D., et al., Cancer Treat. Rep., 69:817–820, 1985; Rios, A., et al., J. Clin. Oncol., 3:506–512, 1985). It is also effective for the treatment of hepatitis C virus (Davis, G. G., et al., N. Engl. J. Med., 321:1501–1506, 1989), and its applicable range as a therapeutic agent is expanding day by day.
The result of cloning IFNα gene from leukocyte has shown that IFNα is encoded by a group of at least 10 different genes. This indicates that the DNA sequences of the genes do not produce one kind of protein but that IFNα is a mixture of subtype proteins having similar structures. Such subtype proteins are named IFNα-1, 2, 3, and so on (Nature, 290:20–26, 1981).
Among the several types of interferons, hIFNα purified from human leukocyte has a molecular weight of 17,500 to 21,000, and a very high native activity of about 2×108 IU/mg protein. In vivo IFNα is a protein consisting of 165 amino acids. It is designated IFNα-2a (SEQ ID NO: 1) in case the 23rd amino acid is lycine, and IFNα-2b (SEQ ID NO : 2) in case the 23rd amino acid is arginine. In the beginning hIFNα was produced by a process using a cell culture method. However, this process is unsuitable for commercialization because of its low productivity of about 250 ug/L.
To solve this problem, processes for recovering a large quantity of interferon from microorganisms by using gene recombinant techniques have been developed and used to date.
The most widely employed is a process using E. coli which produces IFNα consisting of 166 or 167 amino acids according to the characteristics of the E. coli cell. These products have an extra methionine residue added at the N-terminal by the action of the ATG codon existing at the site of initiation codon. However, it has been reported that the additional methionine residue can trigger harmful immune response, in the case of human growth hormone (EP Patent Publication No. 256,843).
In addition, most of the expressed IFNα accumulates in cytoplasm in the form of insoluble inclusion bodies and must be converted into an active form through refolding during a purification process. As such a refolding process is not efficient, IFNα exists partially in a reduced form, or forms an intermolecular disulfide coupling body or a defective disulfide coupling body. It is difficult to remove these by-products, which cause a markedly low yield. In particular, it is extremely difficult to remove undesirable interferon by-products such as misfolded interferons.
Recently, in order to solve the above mentioned problems associated with the production of a foreign protein within a microbial cell, various efforts have been made to develop a method based on efficient secretion of a soluble form of the target protein carrying no extra methionine added to the N-terminal.
In this method, a desired protein is expressed in the form of a fusion protein which carries a signal peptide attached to its N-terminal. When the fusion protein passes through the cell membrane, the signal peptide is removed by an enzyme in E. coli and the desired protein is secreted in a native form.
The secretive production method is more advantageous than the microbial production method in that the amino acid sequence and the higher structure of the produced protein are usually identical to those of the wild-type. However, the yield of a secretive production method is often quite low due to its unsatisfactory efficiencies in both the membrane transport and the subsequent purification process. This is in line with the well-known fact that the yield of a mammalian protein produced in a secretory mode in prokaryotes is much lower than that of a prokaryotic protein produced in the same mode in prokaryotes. Therefore, it has been attempted to develop a more efficient secretory production method. For instance, Korean Patent Publication No. 93-1387 discloses an attempt to mass-produce IFNα using the signal peptide of E. coli alkaline phosphatase, but the yield was very low at 109 IU/L culture medium (10 ug/L culture medium). Therefore, there has been a keen interest in developing a method which is capable of producing soluble IFNα having no additional methionine residue added at the N-terminal, using a microorganism on a large scale.
The present inventors have previously generated a new signal peptide of E. coli thermostable enterotoxin II (Korean Patent Application No. 98-38061 and 99-27418) and found that this new secretory signal peptide can be used for the mass-production of the native form of IFNα. Namely, the present inventors have constructed an expression vector containing a gene obtained by ligating IFNα encoding gene instead of enterotoxin II encoding gene to the modified E. coli secretory signal peptide, and developed a secretory production method of IFNα having a native biological activity via the microbial secretory system by culturing the microorganism transformed with said expression vector.